Early risk assessment of circulating endothelial progenitor cells and plasma stromal cell-derived factor-1 for nondisabling ischemic cerebrovascular events

A total of 186 TIA and minor stroke patients were consecutively collected from June 2016 to June 2018 in the Neurology Department of the Affiliated Hospital of Chongqing Medical University, from both out-patient service and hospitalization. All patients developed morbidity within 1 day. Their ages ranged from 18 to 80 years; 12 were excluded cases, and 21 were shedding cases. The NC group consisted of 15 healthy volunteers who were openly recruited after an official announcement from the hospital during the same time period. Their genders and ages were matched to the other two groups. Finally, 153 patients and 15 healthy volunteers were included in the analysis. Among these 153 patients, there were 83 HR-NICE patients and 70 NHR-NICE patients. Blood samples were taken immediately after admission for TIA and minor stroke patients, and FCM was adopted to measure the amount of circulating EPCs. Using ELISA, the concentrations of serum SDF-1 and VEGF were measured. For the 15 patients in the NC group, only circulating EPCs were extracted. Meanwhile, circulating EPCs were extracted from the first 15 patients in both the HR-NICE and NHR-NICE groups. The MTT method was used to measure the proliferation of EPCs of peripheral blood in all groups, and the Boyden chamber was used to measure the migration of EPCs. NICE refers to ischemic cerebrovascular events that leave no significant disability after morbidity, which includes three types of patients: 1. those who had a TIA; 2. those who had a minor stroke; and 3. those whose symptoms rapidly alleviated and left no significant disability after morbidity. In this research, the definition of HR-NICE is as follows: high-risk TIA (ABCD2 score ≥ 4 points) and minor stroke patients who developed morbidity within 24 h [1]. The definition of NHR-NICE is TIA (ABCD2 score < 4 points). Participants were excluded if they had any history of the following: trauma, blood system diseases, renal insufficiency, inflammation, ulcer, abnormal liver function, malignant tumors, myocardial infarction, angina, atrial fibrillation, recent surgical history, and usage of statins or EPO within 2 months before morbidity. Minor stroke can be defined as NIHSS score ≤ 3; The definition of “no significant disability after morbidity” is as follows: severe symptoms during morbidity but the symptoms alleviated as TIA or minor stroke as in treatment. This research was approved by the Ethics Committee of Yongchuan Hospital of Chongqing Medical University. All patients or their relatives signed an informed consent form.

CD34+/KDR+ were adopted as a marker of circulating EPCs [6]. After admission, 8 mL of antecubital venous blood was extracted and added to a buffer solution with 3.8% heparin. A density gradient centrifugation was used to obtain a monocyte layer and to adjust the density of the cells to 1 × 10⁶/ml. 20 μl of Mouse Anti-Human CD34 monoclonal IgG1 (Southern Biotech Inc., U.S.) was added with a PE mark and 10 μl of Mouse Anti-Human KDR monoclonal IgG1 (R&D Inc., U.S.) was added with an FITC mark. These were then incubated at room temperature without light for 30 min. Next, the samples were centrifuged at 4 °C for 5 min at 1500 rpm/min, and the supernatant was removed and washed twice with PBS. Then, FCM was used to conduct quantitative analysis. The percentage of CD34+/KDR+ cells account for monocyte cells and were used to measure the level of EPCs.

Cultivated monocytes were obtained by density gradient centrifugation in M199 medium (Hyclone, USA) until cell climbing on the 7th day. These were fixed with 2% paraformaldehyde for 15 min and washed in PBS 3 times. Fifty microliters of DIL-acLDL (10 μg/ml) was added then the cells were cultivated at 37 °C in an incubator for 1 h. Next, they were washed twice with PBS, and fixed with 2% paraformaldehyde for 10 min at room temperature. Fifty microliters of FITC-UEA-Lectin (20 μg/ml) was added, and then the cells were cultivated for 2 h at 4 °C without light and washed with PBS three times. The cells were identified by laser confocal microscopy, where FITC-Lectin positive was in green fluorescence, DIL-acLDL positive was in red fluorescence, and the two target cells were yellow, which were the EPCs under differentiation.

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed according to the protocol of the manufacturer to determine the proliferation of EPCs [7]. The density of cultured EPCs was adjusted for 48 h to 1 × 10⁶/ml, and 100 μl of EPCs were inoculated into the culture plate coated with human fibronectin. Then, 20 μl of MTT solution (5 mg/ml) was added to each hole and cultivated for 6 h. The supernatant was removed. Then, 150 μl of dimethyl sulfoxide was added to each hole and oscillated for 10 min. The absorbance was measured at 490 nm by automatic enzyme labeling. For the clonogenic assay, the cells were seeded in 60 mm dishes (1000 cells/plate). Twelve hours after seeding, three groups of cells were allowed to grow until visible colonies appeared. The colonies were stained with 0.01% crystal violet (Sigma, St. Louis, MO) and counted under a microscope.

Cell migration was quantified using a Boyden chamber. Briefly, the density of the EPCs cultured for 48 h was adjusted to 1 × 10⁶/ml. A 50 μl cell suspension was added to the upper chamber of the Boyden chamber. Then, 25 μl of culture medium and VEGF (50 ng/ml) were added to the lower chamber of the Boyden chamber. Cells were cultivated in an incubator for 6 h. Then, the cells were stained at high magnification. Immobile cells were scraped off the membrane, fixed with methanol, Giemsa stained, and counted under a high-power lens.

Three milliliters of antecubital venous blood was extracted and added to a buffer solution with 3.8% heparin. Then, the solution was centrifuged at 4 °C for 15 min at 3000 rpm/min, and the supernatant was collected and kept it in a − 80 °C freezer. ELISA was performed according to the protocol of the manufacturer to determine the concentration of serum SDF-1 and VEGF.

The comparison of clinical data between the NHR-NICE and HR-NICE groups is in Table 1. There were 85 males and 68 females in the 153 TIA and minor stroke cases, whose average age was 59.15 years old. However, patients in the HR-NICE group were elderly and had more hypertension and diabetes (P < 0.01). The laboratory data showed that compared with the NHR-NICE group, patients in HR-NICE had higher levels of peripheral blood triglyceride, total cholesterol, and low-density lipoprotein. Regarding the other variables such as the sampling time, gender, smoking, BMI (Body Mass Index), and HCY (homocysteine), there were no significant differences between the two groups (P > 0.05).

The expression of EPCs, SDF-1 and VEGF in the NHR-NICE group and HR-NICE group is shown in Table 1 above. The data show that the circulating EPCs in the NHR-NICE group accounted for 5.75/000 of monocytes (Fig. 1a), while those in the HR-NICE group accounted for 4.09/000 (Fig. 1b). The mean values of serum SDF-1 in the two groups were 470.30 pg/ml and 755.12 pg/ml and inter-assay CV of 4.1%, respectively. The mean serum VEGF values of the two groups were 70.97 pg/ml and 83.28 pg/ml and inter-assay CV of 3.7%, respectively. In addition, the intra-assay CV of serum SDF in the NHR-NICE group and HR-NICE group were 4.3 and 3.9%, the intra-assay CV of serum VEGF in the two groups were 4.6 and 2.8%. The difference between the two groups is significant (P < 0.01).

Univariate logistic regression analysis screened for possible variables, and the results showed that age, hypertension, diabetes, triglycerides, total cholesterol, low-density lipoprotein, EPCs, SDF-1, VEGF were associated with HR-NICE. However, the risk factors for traditional atherosclerosis include obesity, smoking, and homocysteine, so BMI, smoking, and homocysteine are also included in the multivariate regression analysis. After adjusting for age, BMI, hypertension, diabetes, smoking, triglycerides, total cholesterol, low-density lipoprotein, homocysteine, EPCs, SDF-1, and VEGF for multivariate logistic regression analysis, the results showed the following: age (OR:1.134, 95% CI: 1.030–1.249, P = 0.011), hypertension (OR: 10.798, 95% CI: 2.174–53.622, P = 0.004), diabetes (OR: 11.630, 95% CI: 1.487–90.941, P = 0.019), TC (OR: 2.416, 95% CI: 0.971–6.010, P = 0.058), EPCs (OR: 0.067, 95% CI: 0.023–0.198, P < 0.01), and SDF-1 (OR: 1.007, 95% CI: 1.003–1.011, P < 0.01) are all independent risk factors for HR-NICE (Table 2).

EPC double phagocytosis experiments showed that FITC-Lectin was positive when green fluorescence was present (Fig. 2a), DIL-acLDL was positive when red fluorescence was present (Fig. 2b), and double-labeled cells were yellow for the differentiation of EPCs (Fig. 2c). Peripheral blood was isolated and cultured for 48 h in EPCs. An MTT assay was used to detect the proliferation and migration ability of each group. The results showed that compared with the NC group, the cell proliferation abilities of the HR-NICE group and the NHR-NICE group were inhibited, and the difference was very significant (P < 0.01, Fig. 3a, b). The cell migration ability of the HR-NICE group was weakened (P < 0.01, Fig. 3c, d). The cell migration ability of the NHR-NICE group was weaker than that of the NC group, and the difference was significant (P < 0.05). Compared with the NHR-NICE group, the cell proliferation and migration ability of the HR-NICE group was decreased (P < 0.01).